The specific aims of this project are to generate E. coli clones containing gene replacement mutations in approximately 1,300 protein-coding genes of Tetrahymena thermophila and to transform these clones into the Tetrahymena germline genome to generate knockout strains. The clones and strains will be cryogenically preserved and made freely available to any interested researcher. This resource will greatly facilitate the efforts of an active research community using this model organism and attract new investigators to its study. Tetrahymena has been studied in the laboratory for nearly a century and has contributed to many discoveries of fundamental importance in basic and biomedical research. Prominent areas of investigation include chromatin modification, RNA interference, telomere formation and maintenance, cytoskeletal dynamics, secretion, and DNA replication, amplification and elimination. The availability of whole genome sequence and annotation information has opened up new fields of investigation and attracted new researchers to this model. Tetrahymena has retained many ancestral eukaryotic genetic functions that have been lost in other unicellular model organisms, such as yeast. Many of these genes have human orthologs that are disease- associated. Because Tetrahymena undergoes exclusively homologous gene replacement, many of these genes have been studied by creating knockouts. A unique advantage of Tetrahymena for such analyses is that knockouts of essential genes can be maintained in the transcriptionally silent germline micronucleus and brought into expression by a simple mating. However, the generation of knockout constructs and strains has thus far proceeded on an inefficient, gene-by-gene basis that has hindered the progress of Tetrahymena research and precluded investigation of larger gene families. By optimizing and centralizing this process, we intend to overcome this hurdle for the long-term enhancement of the capabilities of the Tetrahymena research community. A large number of researchers have expressed enthusiasm for this high throughput project and identified numerous knockout targets of particular interest. The knockout strains will be transferred to the Tetrahymena Stock Center and made available to all investigators. The methods developed will serve as a model for future efforts to generate a complete set of Tetrahymena knockout strains. PUBLIC HEALTH RELEVANCE: Research with the simple model organism Tetrahymena has contributed to our understanding of several fundamental processes, such as how chromosome ends replicate;that particular study advanced studies of aging and cancer. Numerous other basic studies with far-reaching relevance to human health would be facilitated by the availability of the proposed freely available resource.